RECENT FINDINGS: (A) STRUCTURAL AND FUNCTIONAL STUDIES OF ORF1p - ORF1p is one of the two L1 encoded proteins essential for retrotransposition. ORF1p binds nucleic acids, acts as a nucleic acid chaperone, and can form multimers via a highly conserved coiled coil domain, a domain that has been repeatedly subject to adaptive evolution. Despite recent progress, the relation between the structural &biochemical properties of ORF1p &its function in retrotransposition is largely unknown. We are addressing this problem using several approaches including analysis of the effects of adaptive evolution. We resuscitated the pre-adapted ORF1p of the L1Pa5 family, ancestor to the adapted ORF1p of the currently active human L1Pa1 family. We call the L1Pa5 protein 555, the modern one, 111, and mosaic versions thereof, 511, 151, and 551. As the coiled-coil domain resides in the amino terminal half of ORF1p, 511, 151 and 551 contain mosaic coiled coil domains. We purified mg amounts of homogeneous 111 ORF1p and found that the human ORF1p is a trimer (like mouse ORF1p, and, as was recently reported, E. coli synthesized human ORF1p), exhibits nucleic acid chaperone activity, and by gel shift binds nucleic acids (of n &#8805;50) with a stoichiometry of 1 per trimer. We are now determining crystallization conditions for the protein. The mosaic coiled coils can form multimers, &where examined (e.g., 555 &111) with each other. The 555, 511, &551 proteins can replace the modern 111 in supporting retrotransposition. However, 151 cannot though it is synthesized and stable in mammalian cells. Only 9 of the 47 amino acid differences between 551 and 111, reside in 151 and changing just three to their modern counterparts restores retrotransposition activity. Thus minimal changes in the coiled coil domain are determinant of ORF1p activity. We are now purifying sufficient amounts of the 555, 151, its re-activated versions, and other modified forms of the 111 ORF1p to correlate their biochemical &structural properties with their activity in retrotransposition. (B) DEVELOPMENT OF A NEW RETROTRANSPOSITION ASSAY - Retrotransposition assays rely on the detection of a DNA copy (reporter gene) of a spliced RNA transcript. RNA splicing ensures that the DNA copy has gone through an RNA intermediate, the hallmark of retrotransposition. Two features of the current retrotransposition assay preclude analysis of the role of 3'L1 RNA structures for this process: (a) RNA splicing occurs via the splicesomal pathway, not normally encountered by L1 RNA. (b) The 2 kb reporter gene obliterates the normal structure of the 3'UTR L1 RNA. We ultimately plan to replace the reporter gene with a 15 bp sequence split by the self-splicing (autocatalytic) intron. Thus, upon synthesis splicing will generate a transcript that differs from a normal L1 transcript by only 15 nucleotides. We will measure retrotransposition events by highly sensitive and specific hybridization to PNAs (amide linked nucleic acid polymers) as has been developed by Dr. Daniel Appella's group. By coupling this reaction with quantitative PCR we can measure both the retrotransposition end product and its intermediates, spliced RNA and cDNA. We have now determined the optimal conditions and PNA sequences for hybridizing both strands of bona fide retrotransposition products from genomic DNA, and are now determining the conditions for measuring the RNA and cDNA intermediates